Calcineurin is a Ser/Thr phosphatase and a primary regulator for several signal transduction cascades, including those involved in immune cell activation, skeletal muscle development, memory formation, and cardiac muscle hypertrophy. The hypothesis for this project is that protein-protein interactions, which determine calcineurin substrate specificity, and oxidative stress, which inactivates calcineurin in vitro, act in conjunction with Ca2+ and calmodulin to ensure proper calcineurin function in intact cells. Although Ca2+ and calmodulin are required for calcineurin activity, other regulatory mechanisms are essential for proper direction of the enzyme and to act as a brake on activity. The relative importance of these mechanisms is unknown and nothing is known about interactions among and integration of the regulatory mechanisms. The objective of this project is to determine how calcineurin targeting proteins direct the enzyme and to what extent oxidants function as an "off switch" in intact cells. To this end constructs for expression of calcineurin proteins as fusions with fluorescent calmodulin indicator proteins (CN/CaM-Ind) will be made. These CN/CaM-Ind will be used in cells with a stably integrated NFAT reporter, which measures calcineurin activity, to determine the cellular localization and calmodulin binding to calcineurin under various conditions. Aims of the project are: 1) To produce and test the CN/CaM-Ind system. 2) To determine the role of the CN-A amino terminus in enzyme activation, CN-A/CN-B interaction and NFAT substrate binding. 3) To determine if calcineurin targeting is Ca2+-dependent and if the VP/IT calcineurin binding domain directs calcineurin activity toward specific residues either by distance or direction. 4) To determine if calcineurin in intact cells is reversibly inhibited by oxidants and if targeting protein binding or calmodulin-dependent activation increase susceptibility to oxidants. Because calcineurin plays such a pivotal role in NFAT signaling and targeting is essential for calcineurin in that signaling cascade, the results of these experiments will enable us to design substrate specific calcineurin inhibitors which would be superior immunosuppressive agents. In addition, the contribution of calcineurin activity to the physiological and pathological responses to oxidative stress will be defined and interventive strategies to minimize the adverse health effects of oxidants will be found.